Mechanism of resistance to cyclopentenyl cytosine (CPE-C) in Molt-4 lymphoblasts

Susan M. Blaney, Jean L. Grem, Frank M. Balis, Diane E. Cole, Peter C. Adamson, David G. Poplack

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Cyclopentenyl cytosine (CPE-C), a carbocyclic analogue of cytidine, has preclinical antineoplastic activity against ara-C resistant murine leukemias and a broad spectrum of human tumor xenografts. CPE-C is a prodrug and requires intracellular phosphorylation to cyclopentenyl cytosine triphosphate (CPE-CTP) which depletes endogenous CTP pools. The initial step in this activation process is catalyzed by uridine/cytidine kinase. We studied the mechanism of resistance to CPE-C in a Molt-4 T-cell leukemia line made resistant to CPE-C (Molt-4R) by culturing it in the continuous presence of increasing concentrations of CPE-C. Using a tetrazolium based colorimetric assay to assess cytotoxicity, the ic90 for the parent Molt-4 cells (Molt-4WT) was 0.5 μM after a 24 hr drug exposure. In contrast, cytotoxicity was not observed at concentrations as high as 1 mM in the Molt-4R cells. Following a brief exposure to 1 μM CPE-C, parent drug could be detected intracellularly in the resistant and sensitive cell lines. However, CPE-CTP formation was reduced markedly in the resistant cell line. Measurement of the activity of anabolic and catabolic enzymes in the Molt-4WT and Molt-4R cells revealed equivalent activities of alkaline and acid phosphatases as well as cytidine and dCMP deaminase but there was a significant reduction in uridine/cytidine kinase activity in Molt-4R cells. Endogenous ribonucleotide pools and CPE-CTP pools were measured in the absence and presence of CPE-C. CTP pools were reduced markedly in Molt-4WT cells following exposure to CPE-C. However, CTP pools in Molt-4R cells exposed to 100 μM CPE-C were two times greater than in the untreated Molt-4WT cells. At high concentrations of CPE-C (10 and 100 μM), Molt-4R cells were able to generate amounts of CPE-CTP equivalent to that seen in Molt-4WT cells exposed to 1 μM CPE-C (a cytotoxic concentration of drug in Molt-4WT cells), but no cytotoxic effect was seen in Molt-4R cells. Therefore, in addition to decreased uridine/cytidine kinase activity, a second mechanism of resistance that is the result of alterations in CTP synthetase activity also appears to be operative. Elucidation of the mechanism of resistance in vitro may provide insight into the mechanism of action of the drug and potential mechanisms of resistance in vivo.

Original languageEnglish (US)
Pages (from-to)1493-1501
Number of pages9
JournalBiochemical Pharmacology
Volume45
Issue number7
DOIs
StatePublished - Apr 6 1993

Fingerprint

cyclopentenyl cytosine
Uridine Kinase
Cytidine Triphosphate
Cytidine
Cytotoxicity
CTP synthetase
Cell Line
Pharmaceutical Preparations
Cells
Cytidine Deaminase

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

Mechanism of resistance to cyclopentenyl cytosine (CPE-C) in Molt-4 lymphoblasts. / Blaney, Susan M.; Grem, Jean L.; Balis, Frank M.; Cole, Diane E.; Adamson, Peter C.; Poplack, David G.

In: Biochemical Pharmacology, Vol. 45, No. 7, 06.04.1993, p. 1493-1501.

Research output: Contribution to journalArticle

Blaney, Susan M. ; Grem, Jean L. ; Balis, Frank M. ; Cole, Diane E. ; Adamson, Peter C. ; Poplack, David G. / Mechanism of resistance to cyclopentenyl cytosine (CPE-C) in Molt-4 lymphoblasts. In: Biochemical Pharmacology. 1993 ; Vol. 45, No. 7. pp. 1493-1501.
@article{449bb1cc1e39415d9feb517e9887ecea,
title = "Mechanism of resistance to cyclopentenyl cytosine (CPE-C) in Molt-4 lymphoblasts",
abstract = "Cyclopentenyl cytosine (CPE-C), a carbocyclic analogue of cytidine, has preclinical antineoplastic activity against ara-C resistant murine leukemias and a broad spectrum of human tumor xenografts. CPE-C is a prodrug and requires intracellular phosphorylation to cyclopentenyl cytosine triphosphate (CPE-CTP) which depletes endogenous CTP pools. The initial step in this activation process is catalyzed by uridine/cytidine kinase. We studied the mechanism of resistance to CPE-C in a Molt-4 T-cell leukemia line made resistant to CPE-C (Molt-4R) by culturing it in the continuous presence of increasing concentrations of CPE-C. Using a tetrazolium based colorimetric assay to assess cytotoxicity, the ic90 for the parent Molt-4 cells (Molt-4WT) was 0.5 μM after a 24 hr drug exposure. In contrast, cytotoxicity was not observed at concentrations as high as 1 mM in the Molt-4R cells. Following a brief exposure to 1 μM CPE-C, parent drug could be detected intracellularly in the resistant and sensitive cell lines. However, CPE-CTP formation was reduced markedly in the resistant cell line. Measurement of the activity of anabolic and catabolic enzymes in the Molt-4WT and Molt-4R cells revealed equivalent activities of alkaline and acid phosphatases as well as cytidine and dCMP deaminase but there was a significant reduction in uridine/cytidine kinase activity in Molt-4R cells. Endogenous ribonucleotide pools and CPE-CTP pools were measured in the absence and presence of CPE-C. CTP pools were reduced markedly in Molt-4WT cells following exposure to CPE-C. However, CTP pools in Molt-4R cells exposed to 100 μM CPE-C were two times greater than in the untreated Molt-4WT cells. At high concentrations of CPE-C (10 and 100 μM), Molt-4R cells were able to generate amounts of CPE-CTP equivalent to that seen in Molt-4WT cells exposed to 1 μM CPE-C (a cytotoxic concentration of drug in Molt-4WT cells), but no cytotoxic effect was seen in Molt-4R cells. Therefore, in addition to decreased uridine/cytidine kinase activity, a second mechanism of resistance that is the result of alterations in CTP synthetase activity also appears to be operative. Elucidation of the mechanism of resistance in vitro may provide insight into the mechanism of action of the drug and potential mechanisms of resistance in vivo.",
author = "Blaney, {Susan M.} and Grem, {Jean L.} and Balis, {Frank M.} and Cole, {Diane E.} and Adamson, {Peter C.} and Poplack, {David G.}",
year = "1993",
month = "4",
day = "6",
doi = "10.1016/0006-2952(93)90050-7",
language = "English (US)",
volume = "45",
pages = "1493--1501",
journal = "Biochemical Pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",
number = "7",

}

TY - JOUR

T1 - Mechanism of resistance to cyclopentenyl cytosine (CPE-C) in Molt-4 lymphoblasts

AU - Blaney, Susan M.

AU - Grem, Jean L.

AU - Balis, Frank M.

AU - Cole, Diane E.

AU - Adamson, Peter C.

AU - Poplack, David G.

PY - 1993/4/6

Y1 - 1993/4/6

N2 - Cyclopentenyl cytosine (CPE-C), a carbocyclic analogue of cytidine, has preclinical antineoplastic activity against ara-C resistant murine leukemias and a broad spectrum of human tumor xenografts. CPE-C is a prodrug and requires intracellular phosphorylation to cyclopentenyl cytosine triphosphate (CPE-CTP) which depletes endogenous CTP pools. The initial step in this activation process is catalyzed by uridine/cytidine kinase. We studied the mechanism of resistance to CPE-C in a Molt-4 T-cell leukemia line made resistant to CPE-C (Molt-4R) by culturing it in the continuous presence of increasing concentrations of CPE-C. Using a tetrazolium based colorimetric assay to assess cytotoxicity, the ic90 for the parent Molt-4 cells (Molt-4WT) was 0.5 μM after a 24 hr drug exposure. In contrast, cytotoxicity was not observed at concentrations as high as 1 mM in the Molt-4R cells. Following a brief exposure to 1 μM CPE-C, parent drug could be detected intracellularly in the resistant and sensitive cell lines. However, CPE-CTP formation was reduced markedly in the resistant cell line. Measurement of the activity of anabolic and catabolic enzymes in the Molt-4WT and Molt-4R cells revealed equivalent activities of alkaline and acid phosphatases as well as cytidine and dCMP deaminase but there was a significant reduction in uridine/cytidine kinase activity in Molt-4R cells. Endogenous ribonucleotide pools and CPE-CTP pools were measured in the absence and presence of CPE-C. CTP pools were reduced markedly in Molt-4WT cells following exposure to CPE-C. However, CTP pools in Molt-4R cells exposed to 100 μM CPE-C were two times greater than in the untreated Molt-4WT cells. At high concentrations of CPE-C (10 and 100 μM), Molt-4R cells were able to generate amounts of CPE-CTP equivalent to that seen in Molt-4WT cells exposed to 1 μM CPE-C (a cytotoxic concentration of drug in Molt-4WT cells), but no cytotoxic effect was seen in Molt-4R cells. Therefore, in addition to decreased uridine/cytidine kinase activity, a second mechanism of resistance that is the result of alterations in CTP synthetase activity also appears to be operative. Elucidation of the mechanism of resistance in vitro may provide insight into the mechanism of action of the drug and potential mechanisms of resistance in vivo.

AB - Cyclopentenyl cytosine (CPE-C), a carbocyclic analogue of cytidine, has preclinical antineoplastic activity against ara-C resistant murine leukemias and a broad spectrum of human tumor xenografts. CPE-C is a prodrug and requires intracellular phosphorylation to cyclopentenyl cytosine triphosphate (CPE-CTP) which depletes endogenous CTP pools. The initial step in this activation process is catalyzed by uridine/cytidine kinase. We studied the mechanism of resistance to CPE-C in a Molt-4 T-cell leukemia line made resistant to CPE-C (Molt-4R) by culturing it in the continuous presence of increasing concentrations of CPE-C. Using a tetrazolium based colorimetric assay to assess cytotoxicity, the ic90 for the parent Molt-4 cells (Molt-4WT) was 0.5 μM after a 24 hr drug exposure. In contrast, cytotoxicity was not observed at concentrations as high as 1 mM in the Molt-4R cells. Following a brief exposure to 1 μM CPE-C, parent drug could be detected intracellularly in the resistant and sensitive cell lines. However, CPE-CTP formation was reduced markedly in the resistant cell line. Measurement of the activity of anabolic and catabolic enzymes in the Molt-4WT and Molt-4R cells revealed equivalent activities of alkaline and acid phosphatases as well as cytidine and dCMP deaminase but there was a significant reduction in uridine/cytidine kinase activity in Molt-4R cells. Endogenous ribonucleotide pools and CPE-CTP pools were measured in the absence and presence of CPE-C. CTP pools were reduced markedly in Molt-4WT cells following exposure to CPE-C. However, CTP pools in Molt-4R cells exposed to 100 μM CPE-C were two times greater than in the untreated Molt-4WT cells. At high concentrations of CPE-C (10 and 100 μM), Molt-4R cells were able to generate amounts of CPE-CTP equivalent to that seen in Molt-4WT cells exposed to 1 μM CPE-C (a cytotoxic concentration of drug in Molt-4WT cells), but no cytotoxic effect was seen in Molt-4R cells. Therefore, in addition to decreased uridine/cytidine kinase activity, a second mechanism of resistance that is the result of alterations in CTP synthetase activity also appears to be operative. Elucidation of the mechanism of resistance in vitro may provide insight into the mechanism of action of the drug and potential mechanisms of resistance in vivo.

UR - http://www.scopus.com/inward/record.url?scp=0027456263&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027456263&partnerID=8YFLogxK

U2 - 10.1016/0006-2952(93)90050-7

DO - 10.1016/0006-2952(93)90050-7

M3 - Article

C2 - 8471071

AN - SCOPUS:0027456263

VL - 45

SP - 1493

EP - 1501

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 7

ER -